The long term goal of this proposal is to establish human pulp and odontoblast cell lines that retain their ability to express tissue- specific gene products of the pulp and odontoblast phenotypes. The cell lines will be established from dental pulp mesenchyme monolayer cultures derived from human pulp explants. Such cell lines are necessary tools for the analysis of the molecular mechanisms controlling gene expression, the process of extracellular matrix mediated biomineralization, odontoblast cytodifferentiation, and mechanisms of reparative or tertiary dentin formation. In addition, such cell lines will be of extreme benefit for evaluation of the cytotoxicity, inflammatory potential, immune responsiveness, and the mutagenicity and carcinogenicity associated with the development of new dental material. Currently, no cell lines exist for human dental pulp mesenchyme cells and therefore progress in understanding the cell biology and biochemistry of pulp and odontoblast cells has been greatly impeded. Such lines would allow the detailed biochemical and molecular studies to be conducted on a stable, homogeneous populations of cells. This proposal is based on the hypothesis that human dental pulp mesenchyme cells maintained in vitro that are transfected with the viral oncogene, Simian Virus (SV) 40 large T antigen, will become immortalized, establishing stable pulp and odontoblast cell lines, which retain the ability to express tissue- specific gene products. To test this hypothesis, two Specific Aims are proposed: 1. To immortalize human dental pulp mesenchyme cells maintained in monolayer cell culture using the viral oncogene SV 40 large T antigen. 2. To characterize the functional properties of all established cell lines and to define the various phenotypes (i.e,, pulp, preodontoblast, mature odontoblast). The established human cell lines will provide valuable tools for future studies regarding the biochemistry regulation and molecular biology of pulp and odontoblasts. The immortalized human pulp and odontoblast cell lines resulting from this work will represent the only human cell lines available for dental mesenchymal tissue. The availability of human cell lines will facilitate the direct application of research in this field to the development of new clinical treatment plans and procedures. This project will also provide the P.I.s with the foundation for future studies aimed at elucidating the biochemical and molecular programs that govern the cytodifferentiation of odontoblasts and formation of tertiary dentin.